Probe for detecting a highly ordered structural site of a single stranded nucleic acid of a gene, and a method and a device for detecting the same

ABSTRACT

The invention provides a probe for detecting a highly ordered structural site of a nucleic acid of a gene by specifically binding with the structural site to generate an electrochemical response. The inventive probe comprises a cyclic ligand containing ferrocenyl group and a DNA threading intercalating moiety, such as 1,4,5,8-tetrasubstituted naphthalene, 9,10-disubstituted anthracene, and 1,5-disubstituted anthraquinone. Current of the cyclic ligand is not observed due to interaction such as stacking or so called charge transfer between ferrocenyl group and the DNA threading intercalating moiety in conventional electrolyte. The binding of the ligand with a highly ordered structural site of a single stranded nucleic acid, where nucleic base inserts between the cavity of cyclic ligand, inhibits the intramolecular interaction of the ligand to convert the ligand into its electrically active form, and as a result, current is observed.

This Application is a divisional of application Ser. No. 09/313,992,filed May 19, 1999, now U.S. Pat. No. 6,294,670.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates to a probe for detecting a highly orderedstructural site of a single stranded nucleic acid of a gene, a methodfor detecting the same using the probe, and a device for detecting thesame.

2. Related Arts

Sensors and sensing techniques have been utilized in every field ofindustries. In particular in biotechnology field, a high-sensitivitysensor system utilizing an enzyme reaction has been established.Recently, importance of gene sensing is increasing in applications suchas gene therapy and gene diagnosis. Up to now, “DNA probe method” hasbeen accepted as such gene sensing method.

A highly ordered structural site of a single stranded nucleic acid is aregion located in a part of high-order structure of a DNA or RNA wherethe bases of the single stranded nucleic acids are not stacked, theregion including a mismatch structure of an oncogenic DNA, a hairpinstructure of a viral RNA and a bulge.

Such DNA probe method is mostly carried out by manual operations.Consequently, it has been demanded a gene sensor other than the DNAprobe type sensor for detecting efficiently a specialized nucleic acidregion of a gene. However, such sensor has not yet been reported orpatented.

The object of the invention is to provide a detection probe fordetecting a highly ordered structural site of a single stranded nucleicacid of a gene, and to provide a detection method and device using suchprobe.

The invention provides a probe for detecting a highly ordered structuralsite of a single stranded nucleic acid of a gene by specifically bindingwith the site to generate an electrochemical response, the probecomprising a cyclic ligand containing ferrocenyl group and a DNAthreading intercalating moiety.

The DNA threading intercalating moiety is a moiety or residual groupderived from a DNA threading intercalating compound. The DNA threadingintercalating compound is a compound which can intercalate or slidebetween adjacent base pairs of a double stranded DNA with its twosubstituents projecting out of the major and minor groovesimultaneously.

The invention also provides a method for detecting a highly orderedstructural site of a single stranded nucleic acid of a gene, the methodcomprising:

-   -   contacting a gene to be detected with the above probe to        generate an electrochemical response; and    -   detecting the electrochemical response.

The inventors successfully developed a cyclic ligand, whose target is ahighly ordered structural site of a single stranded nucleic acid of agene, generating an electrochemical response only when such site ispresent in the gene. The probe may thereby provide a system for sensinga highly ordered structural site of a single stranded nucleic acid of aDNA or RNA with efficiency and high sensitivity. Moreover, in prior DNAprobes, different DNA probes are required and provided responding todifferent target genes. On the contrary, the inventive detection probemay be applied to every gene having a highly ordered structural site ofa single stranded nucleic acid.

The site of a gene is a part of a high-order structure in a DNA or RNA,including a mismatch structure frequently seen in an oncogenic DNA, anRNA hairpin structure of a virus, and a bulge structure. Such structuralsite comprises basically a single stranded structure, which usuallycoexists with a double stranded structure, causing some deformation inthe structural site compared with a common single stranded nucleic acidstructure. If the site structure is consecutive (such as in the case ofa bulge or hairpin structure), it has a unique stacking structureentirely different from that of a common single stranded nucleic acid.For example, the onset of fragile X syndrome is deeply influenced byrepeated hairpin structures. An HIV virus also preserves mismatch,hairpin and bulge structures present in its TAR-RNA and REE RNA, andsuch structures are indispensable for expressing the function of a HIVvirus.

In the invention, ferrocene and naphthalene diimide (naphthalenebis(dicarboximide) or naphthalene-1,8; 4,5-diimide)) are condensed toobtain a cyclic ligand. Manufacturing example of the cyclic ligand isshown in FIG. 1. This representative cyclic ligand is named “cyclicnaphthalenediimideferrocene” (referred to as “CNDIFc” below). Theinventive cyclic ligand comprises ferrocenyl group, two amide bonds orgroups bonded with both ends of ferrocenyl group, naphthalene diimidemoiety, and two connecting groups each connecting each amide bond orgroup and each N-terminal of naphthalene diimide moiety.

The DNA threading intercalating moiety may preferably comprise anaromatic group selected from a group consisting of1,4,5,8-tetrasubstituted naphthalene, 9,10-disubstituted anthracene and1,5-disubstituted anthraquinone. The connecting manner (the positions ofsubstitution) of the aromatic group plays decisive role as to whetherthe aromatic group works as the threading intercalating moiety(threading intercalator).

The cyclic ligand may further comprise two linker moiety each having twoterminal amino groups. Each linker moiety is connected with the DNAthreading intercalating moiety through one of its terminal amino groupsand is connected with the ferrocenyl group through the other of itsterminal amino groups.

The linker moiety may preferably be a residual group of an amine havingtwo terminal amino groups. The amine may preferably comprise anotheramino group and two alkyl groups. Each alkyl group is bonded with eachterminal amino group and another amino group. In this case, the anotheramino group may preferably be piperazinyl group, methylamino group, oramino group, and most preferably piperazinyl group. The alkyl group maypreferably be C1-6 alkyl group and more preferably be ethyl or propylgroup. The amine may most preferably be one of the followings.

1,4-bis(3-aminopropyl)piperazine, 1,1′-bis(3-aminopropyl)methylamine,1,1′-bis(2-aminoethyl)amine, 1,1′-bis(3-aminopropyl)amine, spermine andspermidine.

The DNA threading intercalating moiety may further comprise, in additionto the aromatic group, carbonyl groups, iminomethylene groups (C═NR, Ris hydrogen or an alkyl group), or thiocarbonyl groups (—C═S), throughwhich the intercalating moiety may be bonded with the terminal aminogroups. When the aromatic group is naphthalene, the terminal aminogroups may be bonded through carbonyl groups, iminomethylene groups, orthiocarbonyl groups to 1, 4, 5, 8 positions of naphthalene, to form, forexample, two imide groups or naphthalene-diimide moiety. When thearomatic group is anthracene, the terminal amino groups are bondedthrough carbonyl groups, iminomethylene groups, or thiocarbonyl groupsto 9 and 10 positions of anthracene. When the aromatic group isanthraquinone, the terminal amino groups are bonded through carbonylgroups, iminomethylene groups, or thiocarbonyl groups to 1 and 5positions of anthraquinone.

Ferrocenyl group and the other terminal amino group of each linkermoiety may preferably be bonded through methylene group or carbonylgroup.

In most preferred production process of the ligand, one mole of1,4,5,8-naphthalene tetracarboxylic acid, its monoanhydride ordianhydride is reacted with two moles of a connecting compound havingtwo N-terminals (terminal amino groups) to produce one mole of a diaminobody. This diamino body has naphthalene diimide moiety and twoconnecting groups, each having one N-end (terminal amino group). Thediamino body is then reacted with ferrocene dicarboxylic acid or itsactive ester to provide a cyclic ligand.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a synthesis scheme of a representative example (CNDIFc) of theinventive detection probe, and

FIG. 2 is graphs showing examples of detecting a specific nucleic acidregion, in which graph (a) is a cyclic voltamogram of CNDIFc using aworking electrode made of gold modified with no gene, graph (b) is acyclic voltamogram of CNDIFc using a working electrode made of goldmodified with a hairpin structure DNA, and graph (c) is a cyclicvoltamogram of CNDIFc after hybridizing the above hairpin structure withan oligonucleotide complementary with the hairpin structure DNA.

The invention will be described below in detail. The inventive detectiondevice has a container holding solution containing the cyclic ligand, aworking electrode modified with a gene, and a counter electrode to theworking electrode, wherein the working electrode and counter electrodeare dipped in the solution. The device may preferably be provided with areference electrode.

The working, counter and reference electrodes may preferably made of,for example, gold, glassy carbon, or carbon. A gene to be detected maybe immobilized onto the working electrode by means of a known method.For example, when the working electrode is made of gold, a thiol groupmay be introduced in a gene to bind it onto the working electrode bymeans of a gold-sulfur coordination bond. Such method for binding a genewith the working electrode of gold is described, for example, by B. A.Connolly, in a publication “Nucleic acids Res.” 13, 4484, 1985.Moreover, a gene may be immobilized onto the working electrode made ofglassy carbon by oxidizing the working electrode with potassiumpermanganate to introduce a carboxylic acid on the surface of theworking electrode, and by binding the carboxylic acid with an amino acidconstituting the nucleic acid of the gene. This method is described byKelly M. Mollan and Susan R. Mikkelsen in a publication “AnalyticalChemistry” 65, 2317-2323 (1993).

Preferably, a gene to be detected as such, or after replicating it bymeans of a polymerase chain reaction, is provided with a thiol groupusing mercaptoethanol and1-ethyl-3-(3′-dimethylaminopropyl)-carbodiimide (WSCI), and thenimmobilized onto an electrode of gold by chemisorption. Alternatively, agene to be detected as such, or after replicating it by means of apolymerase chain reaction using 5′-thiolated oligonucleotide as one ofprimers, is immobilized onto a gold electrode by chemisorption. Thisgold electrode functions as the working electrode. The inventiveelectrochemical determination is carried out in a cell containing theworking, counter and reference electrodes in the presence of the cyclicligand. Oxidation and reduction reactions of ferrocenyl group present inthe inventive cyclic ligand induce current, whose amplitude may providean indicator of whether a gene to be detected has a highly orderedstructural site of single stranded nucleic acid or not and/or thecontent of the site. The amplitude of the induced current may bedetected by a means or apparatus such as a cyclic voltamogram, adifferential pulse voltamogram, or a potentiostat.

Phosphate buffered saline and the other salts may be added, in additionto the inventive cyclic ligand, to the solution. The solution may beadjusted as described below so that the inventive ligand specificallybinds with the site and scarcely binds with a double stranded nucleicacid site. That is, the solution may preferably contain 1 to 50 μM(micromole) of the cyclic ligand, 10 to 100 mM of phosphate bufferedsaline, and 10 to 50 mM of a salt, the salt may preferably being a saltof an alkaline metal such as potassium or sodium.

EXAMPLES

The experimental results will be described below.

A cyclic ligand was synthesized according to the scheme of FIG. 1.

(Synthesis of an Amine Body)

2 g of 1,4,5,8-naphthalene tetracarboxylic acid dianhydride (7.45 mmol)and 40 ml of 1,4-(3-aminopropyl)piperazine (190 mmol) are refluxed intetrahydrofuran for 8 hours. After the mixture was cooled to roomtemperature, hexane was added to precipitate crystal, which was thencollected by filtration. The thus obtained crystal was dissolved in aminimum volume of chloroform and recrystallized in ether. The resultingcrystal was then removed, from which ether was vacuum-evaporated. Thethus obtained crystal was dissolved again in chloroform andrecrystallized in hexane. The resulting crystal was collected. Theamount of the crystal was 330 mg and the yield was 52 percent. Thecrystal showed the following characteristics.

red-brown solid melting point 300° C. ¹H-NMR chemical shifts (CDCl₃,ppm) 1.58, 1.95, 2.27-2.52, 2.71, 4.28, 8.75 IR C = 0 1650 cm⁻¹(Synthesis of an Active Ester of 1,1′-ferrocene Dicarbolylic Acid)

0.14 g (0.5 mmol) of 1,1′-ferrocene dicarboxylic acid was dissolved in15 ml of DMF to obtain solution, to which 0.66 g (1.5 mmol) of POBreagent and 0.21 ml (1.5 mmol) of triethylamine dissolved in 5 ml of DMFwere added dropwise. The resultant solution was stirred at roomtemperature for one and a half hour to obtain reagent solutioncontaining an active ester of 1,1′-ferrocene dicarboxylic acid. Thereagent solution was used to a reaction with the above amine body.

(Production of a Cyclic Ligand)

0.35 g (0.5 mmol) of the amine body was dissolved in 300 ml ofchloroform. To the amine body solution, the reagent solution, containingan active ester of ferrocene dicarboxylic acid, was added dropwise whilestirring the amine body solution. The resultant solution was thenstirred at room temperature for 20 hours. The reaction solution wasfiltered to obtain filtrate, which was solidified at a reduced pressureand dissolved in methanol. The methanol solution was developed by silicagel column chromatography using methanol as its developing solvent toobtain a fraction seemingly containing a cyclic ligand. Methanol wasremoved at a reduced pressure from the fraction to provide precipitate,which was then dissolved in chloroform and washed with saturated sodiumcarbonate solution. Chloroform was removed from the solution to obtaincrystal, which was dried at a reduced pressure and collected. 46 mg ofcrystal was obtained and its yield was 10 percent. The crystal showedthe following characteristics.

yellow crystal melting point 300° C. ¹H-NMR chemical shifts (CDCl₃, ppm)1.56, 1.83, 1.99, 2.26, 2.45, 3.27, 4.28, 4.40, 4.43, 6.85, 8.77 IR C═O1791, 1661 cm⁻¹ elemental analysis H(%) C(%) N(%) calculated: 57.23 5.8111.61 measured: 57.23 5.92 10.95 FAB mass spectrum: M + 1 (871.5)(Example of Detection)

FIG. 2 shows detailed results of the detection of a hairpin structurecomposed of (SEQ ID NO.:1) GCGAAAAACGC. A gold electrode modified with ahairpin type DNA (SEQ ID NO.:2) (5′-HS-GCGAAAAACGC-3′ was dipped insolution containing 10 mM of phosphate buffered saline (pH 7.0), 10 mMof KCI and 0.1 mM of CNDIFc. Ag/AgGl standard electrode (referenceelectrode) and a counter electrode of platinum were used to measure acyclic voltamogram. The results were shown in graph (b) in FIG. 2. 1.2μA of a response current was gained at 572 mV. That is, 20 pmol ofhairpin structures provided a response current of 1.2 μA. Further, inthis system, several femtomole of hairpin structure type DNA's weredetectable. A response current at −457 mV was shown responding to thepresence of naphthalene diimide.

Further, the above experiment was carried out using a gold electrodewithout any modification, providing results shown in graph (a). Theabove response current at 572 mV was not observed. The disappearance ofthe response current is reasonable, because ferrocenyl groups andnaphthalene diimide moieties in CNDIFc's form charge transfer complexesin solution. Moreover, the response current corresponding to thepresence of naphthalene diimide shifted to −312 mV and its amplitude wasbelow ½ of that of the response current shown in the graph (b). Suchresults indicates that a charge transfer complex, with a chargetransferred from ferrocene to naphthalene diimide, was formed inCNDIFc's in solution.

Contrary to this, in graph (b), current response at 572 mV was shown.This response may be explained as follows. Due to the interactionbetween CNDIFc and the structural site, bases constituting nucleic acidsin the site are attached or sandwiched in CNDIFc's, inhibiting the aboveintermolecular charge transfer to induce measurable response current at572 mV corresponding to ferrocene.

Further, in the above experiment, the hairpin structure DNA washybridized with an oligonucleotide (SEQ ID NO.:3)(5′-GCGTTTTTCGC-3′)complementary to the hairpin DNA to cancel the hairpin structure site.The results were shown in graph (c), in which current response at 572 mVwas disappeared. The results show that, even when a double strandednucleic acid site coexists with a specific single stranded nucleic acidsite, the specific single stranded nucleic acid region may be detected.Further, the results definitely confirm that CNDIFc specifically bindsto a highly ordered structural site of a single stranded nucleic acidand thereby provides current response on electrodes.

As described above, by contacting the inventive cyclic ligand containingnaphthalene diimide moiety and ferrocenyl group with a working electrodemodified with a gene and measuring its electrochemical behavior orresponse currents by means of a cyclic voltamogram or differential pulsevoltamogram, whether a highly ordered structural site of a singlestranded nucleic acid is present in the gene (a DNA or RNA) or not,and/or the content of the site may be evaluated. Particularly, theinventive detection probe may show high sensitivity, because it providesan electrochemical response due to the inhibition of charge transferbetween ferrocenyl group and naphthalene diimide moiety, only when theprobe binds with the target site.

Although particular embodiments of the invention has been describedabove, it is understood that the embodiments may be modified withoutdeparting from the scope of the invention, which is limited only by theappended claims.

1. A method for detecting a highly ordered structural site of a nucleicacid of a single stranded nucleic acid of a target gene, the methodcomprising: contacting the gene with a probe to generate anelectrochemical response, and detecting the electrochemical response;wherein the probe comprises a cyclic ligand containing a ferrocenylgroup and a DNA threading intercalating moiety.
 2. A device fordetecting a highly ordered structural site of a single stranded nucleicacid of a target gene using a device comprising: a probe, a container, asolution for dissolving the probe, the solution being held in thecontainer, a working electrode modified with the target gene, theworking electrode dipped in the solution in the container, and a counterelectrode dipped in the solution in the container; wherein the probecomprises a cyclic ligand containing a ferrocenyl group and a DNAthreading intercalating moiety.
 3. A method for detecting a highlyordered structural site of a single stranded nucleic acid of a targetgene, the method comprising: contacting the gene with a probe togenerate an electrochemical response; and detecting the electrochemicalresponse, wherein the probe comprises a cyclic ligand containingferrocenyl group and a DNA threading intercalating moiety; wherein thecyclic ligand further comprises two linker moieties each having twoterminal amino groups, and wherein each linker moiety is connected withthe DNA threading intercalating moiety through one of said terminalamino groups, and each linker moiety is connected with the ferrocenylgroup through the other of said terminal amino groups.
 4. A device fordetecting a highly ordered structural site of a single stranded nucleicacid of a target gene using a device comprising: a probe, a container, asolution for dissolving the probe, the solution being held in thecontainer, a working electrode modified with the gene the workingelectrode dipped in the solution in the container, and a counterelectrode dipped in the solution in the container; wherein the probecomprises a cyclic ligand containing ferrocenyl group and a DNAthreading intercalating moiety, wherein the cyclic ligand furthercomprises two linker moieties each having two terminal amino groups, andwherein each linker moiety is connected with the DNA threadingintercalating moiety through one of said terminal amino groups, and eachlinker moiety is connected with the ferrocenyl group through the otherof said terminal amino groups.